General
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variation and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of the steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. All references cited, including patents or patent applications are hereby incorporated by reference. No admission is made that any of the references constitute prior art.
As used herein the term “derived from” shall be taken to indicate that a specific integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Scientific
It is generally accepted that agents such as hormones and growth factors elicit their biological functions by binding to specific recognition sites, or receptors, in the plasma membranes of their target cells. This typically causes a conformational change in the receptor, and triggers secondary cellular responses which may result in the activation or inhibition of intracellular processes. Such agents are often referred to as biological modifiers. They may be categorised into two classes according to their activity. Agents which have a stimulatory activity are termed agonists, and those which inhibit the effect of the original ligand are termed antagonists.
The discovery of biological modifiers that differ in structure from the original ligand may be medically useful. Such compounds may have slightly different spectra of biological activity, allowing them to be used in very specific situations. Compounds that are chemically simpler than the agent which they mimic are potentially produced in greater quantities and at lower costs. Compounds that are more chemically robust than the original ligand may be administered by more convenient means. For example, peptidyl ligands cannot be taken orally as they will be broken down in the digestive tract, whereas a non-peptidyl agonist may potentially be administered by this route.
Insulin is a peptidyl ligand, and the identification of agonists and antagonists thereof has important pharmaceutical applications in the treatment of insulin related ailments.
Insulin is regarded as the most important regulatory hormone involved in maintaining glucose homeostasis. In vivo, insulin is produced in the pancreatic β-cells of the Islets of Langerhans. It is secreted from these cells in response to a wide range of nutrients, hormones and neurotransmitters. However, glucose is considered the most important regulator of insulin release. Following its release insulin is carried systemically to target tissues such as liver, muscle and fat. It promotes the uptake of glucose in the peripheral tissues and inhibits gluconeogenesis in the liver. Insulin also is involved in: promoting the synthesis of glycogen, lipid, and protein; gene transcription and mRNA turnover, and the transport of specific amino acids and ions.
Insulin works by first binding to the insulin receptor on the cell surface of its target tissues. The insulin receptor is a specific transmembrane glycoprotein composed of two α-subunits and two β-subunits linked by disulfide bonds. The α-subunits are located extracellularly and contain the insulin binding domain. The β-subunits of the receptor pass through the plasma membrane and have an intrinsic tyrosine kinase activity associated with their intracellular domain. The X-ray coordinates of part of the tyrosine kinase domain has been resolved (1, 2), however, the coordinates detailing the structure of the α-subunits are not yet available. Some inferences about the structure of some domains of the α-subunit can be made from fibronectin structures of homologous protein domains resolved by NMR spectroscopy and from the X-ray structure of the closely related IGF-1 receptor (3). Most recently, the general quaternary structure of the insulin receptor has been resolved using electron cryomicroscopy (4). These studies indicate that one insulin molecule probably binds and effectively crosslinks a L1-cysteine rich domain of one α-subunit and the L2 domain of the other α-subunit. This viewpoint is supported by models derived from the complex kinetics of insulin binding and the models of receptor oligomerisation described below. Following insulin binding to the α-subunit, the β-subunit is autophosphorylated on specific tyrosine residues and this promotes the tyrosine kinase activity of the receptor.
The interaction of insulin with its receptor leading to receptor activation is a complex, key initial event in insulin action. The kinetics of insulin binding to its receptor have been discussed in a plethora of literature over the past twenty years and many different models have been proposed to explain the interaction. Much debate still exists over which of these proposed models effectively describes the correct binding kinetics.
The simplest model proposes that the insulin receptor population is characterised by a single class of homogeneous, non-interacting binding sites and that insulin action is directly proportional to the fraction of these receptors that are occupied. Other studies confirm a single class of receptors, with reported dissociation constants in the order of 0.5 to 5 nM, depending on the source of insulin receptors and experimental conditions employed (eg. temperature).
An alternate model is based on studies that report curvilinear Scatchard plots and assume multiple classes of binding sites. R. Kahn et al. (5) propose two specific receptor sites; a high affinity-low capacity site and a low affinity-high capacity site. However, in a recent review, B. J. Hammond et al. (6), question the validity of this and suggest that because these two binding sites exist on each receptor, there is no logical reason why they would possess different affinities or more importantly widely differing concentrations.
Another model explaining the non-linear nature of Scatchard plots assumes negative cooperativity. P. De Meyts et al. (7), suggest that the filling of empty receptor sites by unlabelled insulin increases the dissociation of labelled insulin from other sites, leading to curvilinear Scatchard plots. This was interpreted as resulting from site-site interactions. However, E. Helmerhorst (8) demonstrates that insulin enhances its own dissociation even when binding is characterised by linear Scatchard plots. Additionally, R. Pollet et al. (9) recognise that the dissociation rate of insulin is nearly independent of receptor occupancy, with enhanced dissociation of bound 125I insulin by native insulin occurring in certain conditions where binding occupancy is decreased. D. Donner (10) also question the theory of negative cooperativity because correction for insulin degradation products result in linear Scatchard plots, suggesting that heterogeneous distribution of binding may in fact be due to insulin degradation rather than negative cooperative interactions.
Another two-state model, proposed by R. Corin and D. Donner (11) explains the kinetics observed upon insulin/receptor dissociation and is illustrated below: 
The hormone (H) interacts with the receptor (R1) to result in the formation of a complex (HR1), with subsequent rapid dissociation (k−1). Stabilisation of the complex (HR1) is through conversion (k2) to another state, from which hormone dissociation is slow (k−2). R. Corin and D. Donner (11) postulate that increasing occupancy time favours the formation of the second complex at the expense of the first. Thus, this model supports the kinetic data that identifies a constant association rate, but a dissociation rate which decreases with time. As a result of a decreased dissociation rate, the dissociation constant is also reduced, therefore providing an explanation for the presence of receptors of two apparent different affinities. It is not known if the bound complex HR2 may dissociate directly to H and R2 through k−3 and to R1 through k−4.
A study by E. Helmerhorst and C. Yip (12) support this two-state model, with the binding of insulin to rat liver membranes producing Scatchard plots of both a linear and curvilinear nature. Insulin binding to rat liver membranes at temperatures of 4 to 15° C. is characterised by linear Scatchard plots, while higher temperatures result in plots that are distinctly curvilinear. E. Helmerhorst and C. Yip (12) implicate a single class of homogeneous, non-interacting binding sites of low affinity below 15° C. However, at physiological temperatures, two receptor states exist. In reference to the model of R. Corin and D. Donner (11), this suggests that insulin receptors exist primarily in one state at temperatures below 15 degrees, where R1>R2 and k4 is less than k−4. However, as the temperature increases, these values become more similar, leading to increased R2, and a curvilinear Scatchard plot.
Several models are based on the original suggestion of D. B. Donner and K. Yonkers (13), that insulin binding induces a conformational change in its receptor, with a conversion to a more slowly dissociating complex. P. De Meyts (14) and L. Schaffer (15) models are consistent with this notion where the initial binding of insulin to one α-subunit of the insulin receptor is followed by the cross-linking of insulin to a second α-subunit.
Briefly, the model proposes that insulin contains two binding surfaces on each α-subunit which can interact independently with corresponding surfaces on another α-subunit of the receptor. These binding sites, termed al and α2, have differing affinities with α1 having a higher affinity than the α2 region. If insulin binds to the α1 site on one α-subunit, it is suggested that this induces a conformational change in the receptor which ultimately allows the insulin molecule to interact with the α2 site on the second subunit. Consequently, the two α-subunits are cross-linked (homo dimerisation), resulting in high affinity binding between insulin and its receptor and subsequent activation of the receptor. However, although another insulin molecule may potentially bind to the α1 site that is vacant, the previous conformational change prevents it from interacting with the second α2 site, causing this interaction to be one of lower affinity. L. Schaffer (15) suggests that negative interactions between the cross-linked tracer insulin and a native insulin molecule bound at this second α1 site may explain the accelerated dissociation in the presence of high concentrations of insulin. However, B. J. Hammond et al. (6) discount this explanation and propose that the acceleration is in fact due to the destabilisation of the cross-linked formation upon the second insulin molecule binding. At very high concentrations of unlabelled insulin, acceleration of tracer insulin dissociation from receptor is inhibited and is attributed by both P. De Meyts (14) and L. Schaffer (15) to monovalent insulin binding of a third insulin molecule to the vacant low affinity α2 site (B. J. Hammond et al. (6)). Therefore, although the stoichiometry of insulin binding to its receptor can support up to three molecules of insulin bound per α2β2 receptor, the more complex 1:2 and 1:3 complexes occur at supra physiological concentrations of insulin and may not be physiologically relevant.
Another factor that may contribute to the complexity of the interaction between insulin and its receptor is the possible involvement of other non-receptor related molecules. J. T. Harmon et al. (16), propose the presence of a membrane protein affinity regulator of insulin binding in rat liver membranes from ob/ob mice. They report that the effect of this protein is to cause a lowering of insulin affinity for its receptor. In the presence of high salt, high pH or purification of receptors on lectin columns, insulin's apparent affinity for its receptor increases. This effect is reversed by dilution of salt or reduction of pH. A similar effect in human placental plasma membranes has been observed and the apparent increase in affinity that has been determined is due to a two-fold increase in the high affinity component of insulin binding rather than an increase in affinity of either the high or low affinity site. R. A. Kohanski and M. D. Lane (17) also demonstrate the presence of a peripheral membrane glycoprotein which is involved in the modulation of insulin binding, while J. Maturo and M. Hollenberg (18) observe a reduction in binding affinity when purified insulin receptors are supplemented with the glycoprotein eluant from the affinity column used to purify them (also observed in our laboratory). E. R. Mortensen et al (19) demonstrate that mild reduction of adipocyte plasma membranes increases the proportion of high affinity insulin binding which is decreased in the presence of GTP, and is temperature and concentration dependent. Thus, the temperature effect observed by E. Helmerhorst and C. Yip (12) may be attributed to the presence of a G-protein that modulates insulin binding. More recently, the presence of a 66 kDa GTP-binding protein in human placental plasma membrane preparations has been shown to influence the binding kinetics of insulin (20). A decrease in insulin binding to the insulin receptor occurs upon pre-incubation of the insulin receptor-G-protein fraction with GTPγS (a stable analogue of GTP), suggesting an important interaction between the insulin receptor and a G-protein. The presence of such a peripheral membrane protein has not been widely espoused in the literature, however, it may well explain some of the complexities of the binding interaction.
Receptor oligormerisation is a ubiquitous phenomenon among growth factor receptors. It may be induced by monomeric ligands such as EGF to induce conformational changes that result in receptor-receptor interactions, or bivalent ligands (eg human growth hormone (hGH)) that mediate dimerisation of neighbouring receptors. The structure of subclass II receptors eg. insulin and IGF-1, are different because they exist as disulfide linked pairs of dimers in a heterotetrameric structure. In these cases, ligand binding to the receptor induces allosteric interaction between the two αβ halves to activate the receptors.
Recently, through high resolution structural and functional studies the fundamental mechanics of ligand-induced receptor activation is beginning to be elucidated. These studies indicate that oligomerisation of receptor subunits may be all that is required for receptor activation. Moreover, the proposed models explain some of the complex kinetics observed in ligandireceptor binding and dose-response studies of these molecules. For example, a model of homodimerisation of growth hormone receptor explains the unusual bell-shaped dose response curve which is caused by monovalent binding of hGH to each receptor monomer and subsequent prevention of dimerisation.
It is generally accepted that insulin binding induces a conformational change in the insulin receptor that is responsible for receptor activation. A number of studies indicate that this conformational change may occur by a cross-linking mechanism, similar to that described above. Firstly, insulin receptors are activated by a number of anti insulin receptor antibodies, but not by monovalent Fab′ fragments, which implies a receptor cross-linking mechanism of activation similar to EGF receptors. Secondly, the homobifunctional cross-linking agent disuccinimide suberate forms more α2 species in the presence than in the absence of insulin, indicating that insulin draws the α-subunits into close proximity. Thirdly, reduced αβ dimer half receptors, when immobilised on wheat germ agglutinin Sepharose do not phosphorylate in the presence of insulin. However, when they are free to associate in solution, insulin causes phosphorylation of the β-subunits, indicating that cross-linking of receptor αβ halves must occur for receptor activation. Finally, concanavalin A induces insulin receptor activation by cross-linking receptors in intact cells, but when monovalent, only slightly induces receptor activation.
Although the dimeric insulin receptor can bind two (or more) insulin molecules (compared with growth hormone which cross-links two hGH receptors) the first insulin molecule binds more tightly than the second molecule and maybe all that is physiologically relevant for receptor activation. In this respect, insulin receptor activation may well be synonymous with hGH activation of its receptor (or similar cytokine/hormone receptor activation). Indeed, the increased rate of dissociation of tracer insulin in the presence of unlabelled insulin has been explained by analogy to the self-antagonism seen for hGH and its receptors.
Insulin binding to the two receptor α-subunits causes activation of the receptor β-subunit tyrosine kinase activity which leads to the phosphorylation of various substrates of insulin action. This includes the phosphorylation of a family of insulin receptor substrates (IRS) that are believed to be the immediate downstream effector molecules of much of insulin action. At present four members of the IRS family have been discovered. It is believed that these molecules have the potential to interact with, and thereby activate, other downstream signalling molecules, leading to many of the actions of insulin. However, the signalling pathways regulated by the individual substrates may vary.
Given the importance of insulin in maintaining glucose homeostasis then, it is clear that any change in the levels of insulin secreted or the responsiveness of cells to insulin may have significant consequences. Decreased secretion of insulin, or decreased responsiveness of cells to insulin results in Diabetes Mellitus, a group of individual conditions, distinguished by the varying causes of hyperglycemia.
Diabetes is a complex disease with many causative factors. Hyperglycemia is a major characteristic of the disease and over time, especially if poorly controlled, leads to many complications of the disease. These complications include microvascular and macrovascular diseases, retinopathy, neuropathy, stroke, hypertension, heart and kidney disease. Careful control of blood glucose levels is, therefore, a key strategy in treatment of diabetes.
Type 1 diabetes mellitus, or insulin-dependent diabetes mellitus (IDDM) is the more severe form of the disease, and is usually detected before 40 years of age. IDDM results from an autoimmune disorder leading to an inability of the body to produce the insulin needed to help maintain blood glucose levels within a normal range (the roles of insulin and how it works is described in more detail below). The symptoms of IDDM include polyuria, polyphagia, polydipsia, weight loss and drowsiness.
Presently, IDDM patients are absolutely dependent on regular injections of insulin for survival. Over 20 million people worldwide are dependent upon insulin in this manner.
Several different types of human insulin are commercially available for diabetics, ranging from the fast-acting Humulin™BR and Novolin™ to slower acting treatments, such as Protamine-zinc-insulin (PZI), Neutral protamine Hagedorn (NPH) insulin and Lente insulin. Insulin analogues like Humalog (LysPro) with altered properties are also available. Each of these insulin therapies have their advantages and disadvantages and so the choice of insulin therapy should be made by the patient and physician with all information about the patient's lifestyle, physical performance, and drug preferences.
The more common form of diabetes, representing in excess of 90% of all diagnosed cases, is referred to as Type 2 diabetes mellitus or non-insulin dependent diabetes mellitus (NIDDM). NIDDM can be triggered by both genetic and environmental factors and is often found in obese individuals. There are at least two fundamental defects associated with NIDDM. One is an increase in the resistance of cells in peripheral tissue to the presence of insulin. Another is decreased secretion of insulin by the β-cells damaged by long-term, elevated blood glucose levels.
The disruption of the insulin receptor substrate (IRS) signalling system involved in mediating the cellular response to insulin also may play a role in the development of NIDDM. Unlike IDDM, the clinical symptoms of NIDDM are often mild, and the condition may even be asymptomatic. NIDDM patients usually do not depend upon insulin injections for their survival. About half adequately control blood glucose levels through dietary therapy and exercise regimes. The others use various oral hypoglycemia agents such as sulfonylureas, biguanides, and α-glucosidase inhibitors, or insulin, or various combinations of them to help regulate their blood glucose levels. The exact drug prescribed to a patient depends not only on the patient's clinical characteristics, but also the pharmacological properties of the treatment.
The sulfonylureas are the most common oral hypoglycemics and are traditionally used in the treatment of non-obese sufferers of NIDDM. They promote the beta-cells in the Islets of Langerhans of the pancreas to secrete insulin and so they effectively augment glucose-induced insulin secretion.
However, these drugs appear to be responsible for inducing hypoglycemic episodes in patients with the incidence of this apparently increasing when sulfonylureas are used in conjunction with alcohol, drugs which potentiate sulfonylurea action, poor food intake or renal impairment. A weight gain is often associated with sulfonylurea use making these compounds undesirable for the treatment of NIDDM in overweight patients.
The biguanide class of oral hypoglycemic agents increase insulin sensitivity and therefore can be used to lower blood glucose levels in NIDDM. Their mechanism of action is unclear. Phenformin is no longer used to treat NIDDM as it can cause fatal lactic acidosis. Metformin now is the only biguanide in clinical use worldwide. It is most commonly used when dietary therapy is unsuccessfully used to regulate blood glucose levels in obese patients. Metformin may be used in conjunction with sulfonylureas in instances where sulfonylurea therapy alone is inadequate or it may be used in combination with insulin in the treatment of IDDM. However, the adverse side-effects of metformin therapy may include lactic acidosis, nausea, bloating, diarrhoea and abdominal cramping.
α-Glucosidase inhibitors are also used to treat NIDDM as an adjunct to dietary measures or sulfonylureas therapy. These compounds allow carbohydrate in the gut to be processed more effectively by slowing down their absorption from the intestinal tract. Adverse side effects of these compounds include flatulence, diarrhoea and abdominal pain.
The thiazolidinediones are another class of compounds that may ameliorate symptoms of NIDDM. These compounds work by reducing insulin resistance at the sites of insulin action in the muscle and liver. They may be used in combination with insulin or sulfonylurea drugs but are not recommended for the treatment of IDDM. Severe adverse side-effects are rarely observed, however, some NIDDM patients fail to respond to this treatment.
Conversely, a number of clinical conditions are characterised by hyperinsulinism that leads to hypoglycaemia.
Insulin or hypoglycaemic drug overdose are clinical conditions that are often difficult to manage and may require hospitalisation and several days of intensive care. Non-accidental overdose or suicide attempts are fairly rare but often lead to death or profound neurologic impairment. The key symptoms of insulin overdose are hypoglycaemia, hypokalaemia and acid-base imbalance. Sulfonylurea drug overdose predominantly causes hypoglycaemia.
Insulinomas account for about 90% of all pancreatic endocrine tumours. They occur with an incidence of about 0.5 per million population and people of all ages can be affected. Early diagnosis and treatment of insulinomas is essential because of their variable manifestations and potential lethality. These tumours are usually benign but synthesise and secrete insulin autonomously causing spontaneous hypoglycaemia. Symptoms may include deep coma, epilepsy, dizziness, weakness, hunger and epigastric pain.
Congenital hyperinsulinism is the most common cause of severe, persistent hypoglycaemia in infants. It may be familial as up to 20% of affected families have more than one affected child. A defect in beta-cell function is the most likely explanation for the hyperinsulinism that can lead to brain damage and death if not detected early.
Gastric dumping syndrome is encountered in approximately 25-50% of patients following gastric surgery and may persist post-operatively for several months. Early dumping usually involves gastrointestinal and vasomotor complaints. Late dumping predominantly involves vasomotor complaints and is a consequence of a reactive hypoglycaemia resulting from hyperinsulinism and an exaggerated release of glucagon-like peptide-1. The gastric dumping syndrome is infrequently reported in children, but is difficult to diagnose and manage and has significant morbidity.
It is critical that the hypoglycaemia, secondary to the hyperinsulinism and characteristic of clinical conditions like those described above, be managed quickly if death or profound neurologic impairment is to be avoided.
The mainstay of therapy in the management of severe hypoglycaemia is glucose or dextrose infusion. In cases of drug overdose, this may follow gut decontamination. Glucagon is used sometimes but considerable caution must be taken because its success depends on limited hepatic glycogen stores. Surgical intervention is quite successful for many insulinomas where the lesion can be appropriately localised. In the case of congenital hyperinsulinism, partial or complete pancreotectomy is often necessary. Surgical excision of injection sites in cases of massive, non-accidental insulin overdose also is sometimes the preferred option.
Diazoxide is sometimes used to combat hyperinsulinism. It is a potent antihypertensive agent and acts to promote blood glucose level by suppressing insulin secretion from the pancreas. However, this drug is not specific in its treatment of hyperinsulinism and a number of undesirable side effects may follow its use. Hypotension, nausea, vomiting, dizziness, weakness and mild liver damage have been reported with its use. Severe hypoglycaemia also persists in some patients following diazoxide therapy.
Octreotide is a peptide analog of somatostatin and, like diazoxide, one of its actions is to inhibit insulin secretion from the pancreas. Octreotide has shown some promise in treating patients with hyperinsulinism, however, resistance to the drug has been reported in some patients with insulinoma. It has been used with some success in treating gastric dumping syndrome in patients refractory to standard therapy. However, its long term use is limited by side-effects such as diarrhoea and steatorrhoea. Moreover, octreotide may in some instances worsen existing hypoglycaemia by suppressing glucagon and growth hormone in the presence of unresponsive pancreatic hyperinsulinism. Octreotide therapy may also have undesired effects on reducing long term growth in infants.
Thus, as can be deduced from the foregoing discussion, the only drugs available for treating hyperinsulinism with secondary hypoglycaemia, act by suppressing further insulin secretion from the pancreas. No other drugs are presently available.
Antagonists of insulin action would be therapeutically useful agents for treating a range of diseases or clinical conditions involving hyperinsulinism and hypoglycaemia. They would work by directly competing with insulin for binding to the insulin receptor (the first step of insulin action) and would thereby counter the effects of hyperinsulinism. Their effect would be to reduce the hypoglycaemic action of insulin evident in clinical conditions described above.
In view of the foregoing, it will be appreciated that there is a current on going need for biological modifiers that are capable of mimicing insulin activity.